Two mutagens isolated from fried-beef patties were compared to a series of synthetic structural isomers of 2-aminodimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-aminotrimethylimidao[4,5-f]quinoxaline (DiMeIQx). Comparison by NMR spectrometry and HPLC coelution showed that one beef mutagen (molecular weight of 213) was identical to the 8-MeIQx isomer not the 7-Me isomer. Another quinoxaline beef mutagen, having 3 methyl groups (molecular weight of 227), had an NMR spectrum different from the 5,8- or 7,8-DiMeIQx isomers, but not clearly distinguishable from the 4,8- or 4,7-DiMeIQx isomers. The HPLC separation of the DiMeIQx isomers and subsequent addition of the beef mutagen showed the beef-derived compound to coelute with the 4,8-DiMeIQx and not with the 4,7-DiMeIQx. The number and position of methyl groups was responsible for a 7-fold range of mutagenic response in the Ames/Salmonella assay. In conclusion, the major quinoxaline mutagens isolated from fried beef were identified as 8-MeIQx and 4,8-DiMeIQx isomers.